department of pharmacology

Evidence is increasing that lack of reproducibility, whatever the cause, is a systemic problem in biomedical science. While institutions like the NIH and concerned journal editors are making efforts to implement more stringent requirements for rigorous and reproducible research, scientists themselves must make conscious efforts to avoid common pitfalls of scientific research. Here at Emory, several scientists are making greater efforts to push forward to improve scientific research and combat what is being called “the reproducibility crisis.”

In 2012, C. Glenn Begley, then a scientist with the pharmaceutical company Amgen, published a commentary in Nature on his growing concern for the reproducibility of preclinical research. Begley and his colleagues had attempted to replicate 53 published studies they identified as relevant to their own research into potential pharmaceuticals. They found that only 6 of the 53 publications could be replicated; even with help from the original authors. Similar studies have consistently found that greater than 50 percent of published studies could not be replicated. This sparked a period of great concern and questioning for scientists. It seemed to Begley and others that experimenter bias, carelessness, poor understanding of statistics, and the career-dependent scramble to publish contributes to a misuse of the scientific method. These factors contribute to what is now called the reproducibility crisis. In April 2017, Richard Harris published Rigor Mortis, a survey of the problem in preclinical research, which has kept the conversation going and left many wondering what the best solution to these issues could be. To combat the reproducibility crisis, Harris argues that funding agencies, journal editors and reviewers, research institutions, and scientists themselves all have a role to play.

Emory University School of Medicine researchers have developed tools that enable them to detect small proteins called granulins for the first time inside cells. Granulins are of interest to neuroscientists because mutations in the granulin gene cause frontotemporal dementia (FTD). However, the functions of granulins were previously unclear.

FTD is an incurable neurodegenerative disease and the most common type of dementia in people younger than 60. Genetic variants in the granulin gene are also a risk factor for Alzheimer’s disease and Parkinson’s disease, suggesting this discovery may have therapeutic potential for a broad spectrum of age-related neurodegenerative diseases.

The results were published August 9 by the journal eNeuro (open access).

Thomas Kukar, PhD

Some neuroscientists believed that granulins were made outside cells, and even could be toxic under certain conditions. But with the newly identified tools, the Emory researchers can now see granulins inside cells within lysosomes, which are critical garbage disposal and recycling centers. The researchers now propose that granulins have important jobs in the lysosome that are necessary to maintain brain health, suppress neuroinflammation, and prevent neurodegeneration.

Problems with lysosomes appear in several neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

“A lysosomal function for granulins is exciting and novel. We believe it may provide an explanation why decreased levels of granulins are linked to multiple neurodegenerative diseases, ranging from frontotemporal dementia to Alzheimer’s,” says senior author Thomas Kukar, PhD, assistant professor of pharmacology and neurology and the Emory University Center for Neurodegenerative Disease. Read more

Anyone familiar with Alzheimer’s disease research can say what a challenge drug development has been. In Emory’s Department of Pharmacology, Thota Ganesh is focusing on an anti-inflammatory approach. Ganesh’s work has been supported by the Alzheimer’s Drug Discovery Foundation and more recently by a five-year, $3.6 million grant from the National Institute on Aging.

EP2 is one of the four receptors for prostaglandin E2, a hormone involved in processes such as fever, childbirth, digestion and blood pressure regulation. Before Ganesh and colleagues from the Emory Chemical Biology Discovery Center started looking for them, chemicals that could block EP2 selectively were not available.

Their idea is: blocking EP2 is a better strategy than the more general approach of going after prostaglandins, the targets for non-steroid anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and celecoxib (Celebrex). Read more

In Emory’s Department of Pharmacology, the Traynelis and Yuan labs have been harvesting the vast amounts of information now available from public genome databases, to better understand how changes in the NMDA receptor genes relate to function. (Take a “deeper dive” into their November 2016 publication on this topic here.)

Their recent paper in PLOS Genetics focuses on a particular region in the NMDA receptor, called the pre-M1 helix (see figure). It also includes experiments on whether drugs now used for Alzheimer’s disease, such as memantine, could be repurposed to have beneficial effects for patients with certain mutations. The in vitro data reported here could inform clinical use. Read more

Inflammation in the brain is a feature of several neurological diseases, ranging from Parkinson’s and Alzheimer’s to epilepsy. Nick Varvel, a postdoc with Ray Dingledine’s lab at Emory, was recently presenting his research and showed some photos illustrating the phenomenon of brain inflammation in status epilepticus (prolonged life-threatening seizures).

Both markers, CX3CR1 (green) and CCR2 (red), are chemokine receptors. Green fluorescent protein is selectively produced in microglia, which settle in the brain before birth and are thought to have important housekeeping/maintenance functions.

Monocytes, a distinct type of cell that is not usually in the brain in large numbers, are lit up red. Monocytes rush into the brain in status epilepticus, and in traumatic brain injury, hemorrhagic stroke and West Nile virus encephalitis, to name some other conditions where brain inflammation is also seen.

In the PNAS paper, Varvel and his colleagues include a cautionary note about using these mice for studying situations of more prolonged brain inflammation, such as neurodegenerative diseases: the monocytes may turn down production of the red protein over time, so it’s hard to tell if they’re still in the brain after several days.

Targeting CCR2 – good or bad? Depends on the disease model

The researchers make the case that “inhibiting brain invasion of CCR2+ monocytes could represent a viable method for alleviating several deleterious consequences of status epilepticus.” Read more

Last week, Lab Land noticed similarities between two independent lines of research from the Escayg and Traynelis/Yuan labs at Emory. Both were published recently and deal with rare forms of genetic epilepsy, in which molecular understanding of the cause leads to individualized treatment, albeit with limited benefit.

Both conditions are linked to an excess of neuronal excitation, and both can be addressed using medications that have also been tested for Alzheimer’s. A critical difference is that memantine is FDA-approved for Alzheimer’s, but huperzine A is not.

The study of human genetics has often focused on mutations that cause disease. When it comes to genetic variations in healthy people, scientists knew they were out there, but didn’t have a full picture of their extent. That is changing with the emergence of resources such as the Exome Aggregation Consortium or ExAC, which combines sequences for the protein-coding parts of the genome from more than 60,000 people into a database that continues to expand.

Rare mutations in the NMDA receptor genes cause epilepsy (GRIN2A) or intellectual disability (GRIN2B). Shown in blue are agonist binding domains of the receptors, where several disease-causing mutations can be found.

At Emory, the labs of Stephen Traynelis and Hongjie Yuan have published an analysis of ExAC data, focusing on the genes encoding two NMDA receptor subunits, GRIN2A and GRIN2B. These receptors are central to signaling between brain cells, and rare mutations in the corresponding genes cause epilepsy (GRIN2A) or intellectual disability (GRIN2B). GRIN2B mutations have also been linked with autism spectrum disorder.

Steve Traynelis and Hongjie Yuan

The new paper in the American Journal of Human Geneticsmakes a deep dive into ExAC data to explore the link between normal variation in the healthy population and regions of the proteins that harbor disease-causing mutations.

In addition, the paper provides a detailed look at how 25 mutations that were identified in individuals with neurologic disease actually affect the receptors. For some patients, this insight could potentially guide anticonvulsant treatment with a repurposed Alzheimer’s medication. Also included are three new mutations from patients identified by whole exome sequencing, one in GRIN2A and two in GRIN2B.

“This is one of the first analyses like this, where we’re mapping the spectrum of variation in a gene onto the structure of the corresponding protein,” says Traynelis, PhD, professor of pharmacology at Emory University School of Medicine. “We’re able to see that the disease mutations cluster where variation among the healthy population disappears.”

Postdoctoral fellow Sharon Swanger, PhD is first author of the paper, and Yuan, MD, PhD, assistant professor of pharmacology, is co-senior author.

It’s not always obvious, looking at the sequence of a given mutation, how it’s going to affect NMDA receptor function. Only introducing the altered gene into cells and studying protein function in the lab provides that information, Traynelis says.

NMDA receptors are complicated machines: mutations can affect how well they bind their ligands (glutamate and glycine), how they open and shut, or how they are processed onto the cell surface. On top of that complexity, mutations that make the receptors either stronger or weaker can both lead the brain into difficulty; within each gene, both types of mutation are associated with similar disorders. With some GRIN2A mutations, the functional changes identified in the lab were quite strong, but the effect on the brain was less dramatic (mild intellectual disability or speech disorder), suggesting that other genetic factors contribute to outcomes.

Memantine is an NMDA receptor antagonist, aimed at counteracting the overactivation of the receptor caused by the mutation. Memantine has also been used to treat children with epilepsy associated with mutations in the related GRIN2D gene. However, memantine doesn’t work on all activating mutations, and could have effects on the unmutated NMDA receptors in the brain as well. Traynelis reports that his clinical colleagues are developing guidelines for physicians on the use of memantine for children with GRIN gene mutations.

This study and related investigations were supported by funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD082373), the National Institute of Neurological Disorders and Stroke (R24NS092989), the Atlanta Clinical & Translational Science Institute (UL1TR000454), and CURE Epilepsy: Citizens United for Research in Epilepsy.

It’s sweet, it’s safe, and it looks like it could save neurons. What is it? Trehalose.

Trehalose is a natural sugar.

This natural sugar is used in the food industry as a preservative and flavor enhancer (it’s in Taco Bell’s meat filling). And curiously, medical researchers keep running into trehalose when they’re looking for ways to fight neurodegenerative diseases.

A recent example from Emory’s Department of Pharmacology: Chris Holler, Thomas Kukar and colleagues were looking for drugs that might boost human cells’ production of progranulin (PGRN), a growth factor that keeps neurons healthy. Mutations in the progranulin gene are a common cause of frontotemporal dementia.

The Emory scientists discovered two leads: a class of compounds called mTOR inhibitors — the transplant drug rapamycin is one — and trehalose. The team decided to concentrate on trehalose because it increased PGRN levels in neuronal and non-neuronal cell types, unlike the mTOR inhibitors. Their results were published at the end of June in Molecular Neurodegeneration.

The team confirmed their findings by examining the effects of trehalose on cells derived from patients with progranulin mutations. This paper is the first to include results from Emory’s Laboratory of Translational Cell Biology, which was established in 2012 to facilitate this type of “disease in a dish” approach. Cell biologists Charles Easley, Wilfried Rossoll and Gary Bassell from the LTCB, and neurologists Chad Hales and William Hu from the Center for Neurodegenerative Disease are co-authors.

Accompanying Kai Kupferschmidtâ€™s July 3 feature in Science, which discusses a current revival of clinical research on hallucinogens such as LSD and psilocybin, was a curious historical photo. The 1955 copyrightedÂ photo depicts pharmacologist Harry Williams squirting LSD into the mouth of Carl Pfeiffer, chair of pharmacology at Emory during the 1950â€™s. Read more

It’s not sleep apnea. It’s not narcolepsy. Hypersomnia is a different kind of sleep disorder. There’s even an “apples and oranges” T-shirt (see below) that makes that point.

This weekend, your correspondent attended a patient-organized Living with Hypersomnia conference. One of the main purposes of the conference was to update sufferers and supporters on the state of research at Emory and elsewhere, but there was also a lot of community building — hence the T-shirts.

The story of how sleep took over one young lawyer’s life, and how her life was then transformed by flumazenil, a scarce antidote to sleeping pills she was not taking, has received plenty of attention.

Now an increasing number of people are emerging who have a condition similar to Anna Sumner’s, and several questions need answers. Read more